Minimized sample consumption for time-resolved serial crystallography applied to the redox cycle of human NQO1
Sample consumption for serial femtosecond crystallography with X-ray free electron lasers remains a major limitation preventing broader use in macromolecular crystallography. This drawback is exacerbated in time-resolved (TR) experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. To reduce this limitation, we demonstrate a segmented droplet generation strategy coupled to a mix-and-inject approach for TR studies at the European XFEL. The injector produces synchronized droplet trains that enable stable and reproducible injection of protein crystal slurries at significantly reduced flow rates. Using the human flavoenzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a test system, we collected diffraction data after mixing with NADH at 0.3 s and 1.2 s delays. The segmented injection approach achieved up to 97% reduction in sample consumption compared with continuous-flow injection while maintaining data quality suitable for TR crystallography. Reproducible electron density features consistent with low-occupancy NADH binding illustrate both the feasibility and the current limits of studying dynamic redox enzymes using this approach. This work establishes segmented droplet generation as a sample-efficient and XFEL-compatible method for future time-resolved serial crystallography experiments.
Authors
- Raphaël de Wijn (ORCID: https://orcid.org/0000-0002-8342-1008)
- Christina Schmidt (ORCID: https://orcid.org/0000-0002-6662-1294)
- Mohammad Vakili (ORCID: https://orcid.org/0000-0001-9776-0177)
- S. Şen (ORCID: https://orcid.org/0009-0006-1025-2430)
- Jayanath Koliyadu (ORCID: https://orcid.org/0000-0002-0245-3842)
- Dmitrii Zabelskii (ORCID: https://orcid.org/0000-0001-6906-975X)
- Joachim Schulz (ORCID: https://orcid.org/0000-0002-9310-8832)
- Johan Bielecki (ORCID: https://orcid.org/0000-0002-3012-603X)
- Alice Grieco
- Adil Ansari
- Gihan Ketawala (ORCID: https://orcid.org/0000-0002-9436-3536)
- Abhik Manna (ORCID: https://orcid.org/0000-0001-8681-6702)
- T. Sato
- Abhisakh Sarma (ORCID: https://orcid.org/0000-0002-0785-8902)
- R. Bean
- Ángel L. Pey (ORCID: https://orcid.org/0000-0001-7706-3243)
- Alexandra Ros (ORCID: https://orcid.org/0000-0001-7709-8331)
- Raimund Fromme (ORCID: https://orcid.org/0000-0003-4835-1080)
- Richard A. Kirian (ORCID: https://orcid.org/0000-0001-7197-3086)
- Thomas D. Grant
- Katerina Dörner (ORCID: https://orcid.org/0000-0003-1072-5905)
- Isabel Quereda-Moraleda
- A. Round
- Samira Mahmud
- Petra Fromme
- Roberto C. Alvarez (ORCID: https://orcid.org/0000-0003-1960-849X)
- Faisal H. M. Koua
- Domin Koh
- Mukul Sonker
- Romain Letrun
- Jose Manuel Martin-Garcia
- Konstantinos Karpos
- Diandra Doppler
- Milagros Medina
- Sabine Botha
- Chan Kim
- Hung Le
- Huijong Han
Institutions
- Consejo Superior de Investigaciones Científicas (ES)
- Universität Hamburg (DE)
- Universidad de Granada (ES)
- La Trobe University (AU)
- Universidad de Zaragoza (ES)
- Deutsches Elektronen-Synchrotron DESY (DE)
- Diamond Light Source (GB)
- National Research Council (LK)
- Center for Free-Electron Laser Science (DE)
- Arizona State University (US)
- University at Buffalo, State University of New York (US)
- European X-Ray Free-Electron Laser (DE)
Publication Details
- Journal
- Communications Chemistry
- Published
- 2026-01-29
- DOI
- https://doi.org/10.1038/s42004-026-01908-9
- Primary Topic
- Enzyme Structure and Function
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Science Foundation
- Arizona State University
- Comunidad de Madrid
- European Commission
- Eyring Materials Center, Arizona State University
- Agencia Estatal de Investigación
- Division of Electrical, Communications and Cyber Systems